OBSERVATIONS IMPLICATING AN EXTRACELLULAR ENZYMIC MECHANISM OF CONTROL OF BONE MORPHOGENESIS

Abstract

Data on physicochemical conditions leading to loss of the bone morphogenetic property of bone matrix in neutral buffer solutions support the concept of an enzymic control mechanism better than a chemical blocking reaction or denaturation. The loss is associated with release of 35S-labeled constituents and not prevented by ε-amino caproic acid, an inhibitor of cathepsins. The loss is also associated with release of 35S-cysteine-labeled protein; about 60% of the yield is sustained by the addition of only 3 mmoles/liter of iodoacetic acid. A latent period of about 12 hr, decreased by extraction of bone matrix with CaCl2, is characterized by release of protein polysaccharide and other noncollagenous proteins. Release of sialic acid from the bone matrix by neuraminidase at pH 7.4 has no effect upon bone yield. At 2°C, Tris-HCl buffer or ethylenediaminetetraacetic acid extracts noncollagenous proteins without loss of bone yield; at 37°C, pH 7.4, these solutions also activate endogenous enzymes and reduce bone yield. The component of bone matrix responsible for reduction in bone yield is separable from bone matrix by extraction with phosphate buffer, by catheptic digestion of bone matrix in acidic buffer solutions, by sequential chemical extraction of noncollagenous proteins with cold slightly acidic salt solutions or by extraction-denaturation with chloroform-methanol. Detergents neither extinguish nor denature the morphogenetic property but some solubilize or extract degradative enzymes; hexodecyl trimethyl ammonium bromide, at pH 5.0, is positively charged and extracts hydrophobic proteins, including part of the bone morphogenetic property. A special selection of sulfhydryl chemical inhibitors remarkably different from the selection inhibiting known enzymes preserves the bone morphogenetic property of bone matrix; p-chloromercuribenzoate preservation is reversible by chemical reactions with cysteine. Reduction in bone yield in phosphate buffer is not attributable to a chemical block because chloroform-methanol extraction of the agent does not restore bone yield and is not attributable to denaturation because bone yield sustained by p-chloromercuribenzoate is lost by chemical reactions with cysteine. An hypothetical insoluble bone morphogenetic protein (BMP) firmly bound to collagen is degraded by a soluble neutral proteinase (BMPase). Digestion of the hypothetical BMP occurs without loss of the 640-A electron micrographic image of bone collagen, resembles tryptic digestion and is more selective as well as physiologic in action.